Polymerase chain reaction (PCR) is a gene amplification method with a simple process, high sensitivity and excellent specificity. Generally, the process is comprised of 20-50 cycles each having three steps: denaturation, annealing and extension. The target nucleic acid can be amplified to millions of times its original concentration in 1.5-3 h. In recent years, real-time PCR amplification has made gel electrophoresis of PCR products unnecessary, thus the post-manipulation of PCR products is not needed, and opportunities for PCR contamination are prevented. Because of these advantages, real-time PCR is used more and more widely. Originally, real-time PCR is used with fluorescent dyes, such as SYBR GREEN, EVE GREEN etc., to detect the PCR product. More recently, nucleic acid probes with fluorescent labels are applied in real-time PCR, such as Taqman probe, molecular beacon, fluorescence resonance energy transform probe, scorpion primers, probes etc. used in 5′-exonuclease technology, Although PCR based on fluorescent dyes is simple, it can not identify the nonspecific amplification, especially the nonspecific amplification caused by primer dimers, so it has many limitations in application. A probe provides a second identification step for the amplification products, therefore the nonspecific amplification is prevented, so its result is more reliable. But the probe has disadvantages such as complex design and high synthetic cost etc. The shortcomings of the two methods are based on the fact that they can not prevent nonspecific amplification.
Rare mutations refer to mutant alleles that are present at a relatively low concentration, or copy number, compared to normal alleles. It especially refers to genes with a single base mutation. Generally, the ratio of mutation gene to wild-type gene is less than 1/1000, for example, trace embryo mutant gene contained in the blood of pregnant woman, or tumor cell-derived mutant DNA contained in blood or tumor tissue of a cancer. The detection of these rare mutations can be a challenge for the detection technologies of the prior art.
The key to successful PCR applied to mutation detection is to design a pair of primers with high precision and complementary to target gene DNA or RNA. Amplification Refractory Mutation System, ARMS was invented in 1989, it is also called Allele-specific PCR (AS-PCR) or PCR with Sequence Specific Primers (PCR-SSP). It has the widest use of any mutation detection method. The basic idea is to design two forward ARMS primers, which have a common reverse primer to form a PCR system. The specific base of allele gene is set in the 3′ end of the primer, this is because the Taq DNA polymerase does not have 3′-5′ exonuclease proofreading activity, in PCR, the specific base in the 3′ end of the primers combine to the sites of wild-type or mutation allele gene; if the base is mismatched, the DNA extension reaction will fail due to the inability to form the 3′-5′-phosphodiester linkage. However, the base of 3′end of ARMS primers has limited ability differentiate between different mismatchs, thus they have limited ability to differentiate some mutations.